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Article: Caged nicotinic acid adenine dinucleotide phosphate. Synthesis and use

TitleCaged nicotinic acid adenine dinucleotide phosphate. Synthesis and use
Authors
Issue Date1997
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
Citation
Journal Of Biological Chemistry, 1997, v. 272 n. 7, p. 4172-4178 How to Cite?
AbstractNicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP with Ca2+ mobilizing activity. The Ca2+ release mechanism activated by NAADp as well as the Ca2+ stores that it acts on are different from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3) (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). In order to demonstrate unambiguously that NAADP can mobilize Ca2+ stores in live cells, a caged analog was synthesized by reacting NAADP with 1-(2- nitrophenyl)di-azoethane. Anion exchange high pressure liquid chromatography (HPLC) was used to purify one particular caged form from the mixture of products. Phosphate analyses following specific enzymatic cleavage indicate that the caging group is on the 2'-phosphate. This is confirmed by 31P NMR spectroscopy, showing that the 2'-phosphate of the caged compound exhibits an altered chemical shift of -2.6 ppm as compared with 2.3 ppm determined for the 2'-phosphate of NAADP. Caged NAADP had no Ca2+ releasing activity at a concentration as high as 1 μM when tested on sea urchin egg microsomes. After photolysis, it released Ca2+, was effective in nanomolar range, and was indistinguishable from authentic NAADP. The regeneration of NAADP after photolysis was also confirmed by HPLC analyses. The analog is particularly susceptible to UV and can be efficiently photolyzed using a spectrofluorimeter. To demonstrate its utility in live cells, caged NAADP was microinjected into sea urchin eggs. Photolysis effectively regenerated NAADP and activated Ca2+ oscillations in the eggs. Removal of external Ca2+ did not prevent the Ca2+ oscillations but only delayed the second Ca2+ peak by about 45 s, indicating that the oscillations are due to release from internal stores and not caused by Ca2+ influx. A mechanism based on sensitization of the Ca2+ release by Ca2+ loading is proposed to account for the Ca2+ oscillation observed.
Persistent Identifierhttp://hdl.handle.net/10722/171456
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
References

 

DC FieldValueLanguage
dc.contributor.authorLee, HCen_US
dc.contributor.authorAarhus, Ren_US
dc.contributor.authorGee, KRen_US
dc.contributor.authorKestner, Ten_US
dc.date.accessioned2012-10-30T06:15:16Z-
dc.date.available2012-10-30T06:15:16Z-
dc.date.issued1997en_US
dc.identifier.citationJournal Of Biological Chemistry, 1997, v. 272 n. 7, p. 4172-4178en_US
dc.identifier.issn0021-9258en_US
dc.identifier.urihttp://hdl.handle.net/10722/171456-
dc.description.abstractNicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP with Ca2+ mobilizing activity. The Ca2+ release mechanism activated by NAADp as well as the Ca2+ stores that it acts on are different from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3) (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). In order to demonstrate unambiguously that NAADP can mobilize Ca2+ stores in live cells, a caged analog was synthesized by reacting NAADP with 1-(2- nitrophenyl)di-azoethane. Anion exchange high pressure liquid chromatography (HPLC) was used to purify one particular caged form from the mixture of products. Phosphate analyses following specific enzymatic cleavage indicate that the caging group is on the 2'-phosphate. This is confirmed by 31P NMR spectroscopy, showing that the 2'-phosphate of the caged compound exhibits an altered chemical shift of -2.6 ppm as compared with 2.3 ppm determined for the 2'-phosphate of NAADP. Caged NAADP had no Ca2+ releasing activity at a concentration as high as 1 μM when tested on sea urchin egg microsomes. After photolysis, it released Ca2+, was effective in nanomolar range, and was indistinguishable from authentic NAADP. The regeneration of NAADP after photolysis was also confirmed by HPLC analyses. The analog is particularly susceptible to UV and can be efficiently photolyzed using a spectrofluorimeter. To demonstrate its utility in live cells, caged NAADP was microinjected into sea urchin eggs. Photolysis effectively regenerated NAADP and activated Ca2+ oscillations in the eggs. Removal of external Ca2+ did not prevent the Ca2+ oscillations but only delayed the second Ca2+ peak by about 45 s, indicating that the oscillations are due to release from internal stores and not caused by Ca2+ influx. A mechanism based on sensitization of the Ca2+ release by Ca2+ loading is proposed to account for the Ca2+ oscillation observed.en_US
dc.languageengen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_US
dc.relation.ispartofJournal of Biological Chemistryen_US
dc.subject.meshAnimalsen_US
dc.subject.meshCalcium - Metabolismen_US
dc.subject.meshChromatography, High Pressure Liquiden_US
dc.subject.meshChromatography, Ion Exchangeen_US
dc.subject.meshMicrosomes - Drug Effects - Metabolismen_US
dc.subject.meshNadp - Analogs & Derivatives - Chemical Synthesis - Isolation & Purification - Pharmacologyen_US
dc.subject.meshOvum - Drug Effects - Metabolismen_US
dc.subject.meshSea Urchinsen_US
dc.titleCaged nicotinic acid adenine dinucleotide phosphate. Synthesis and useen_US
dc.typeArticleen_US
dc.identifier.emailLee, HC:leehc@hku.hken_US
dc.identifier.authorityLee, HC=rp00545en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1074/jbc.272.7.4172en_US
dc.identifier.pmid9020130-
dc.identifier.scopuseid_2-s2.0-0001318493en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0001318493&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume272en_US
dc.identifier.issue7en_US
dc.identifier.spage4172en_US
dc.identifier.epage4178en_US
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridLee, HC=26642959100en_US
dc.identifier.scopusauthoridAarhus, R=6701339421en_US
dc.identifier.scopusauthoridGee, KR=7101946977en_US
dc.identifier.scopusauthoridKestner, T=7801652533en_US

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